Our long-term goal is to understand the critical steps of hTERT gene regulation during development. The hTERT gene, which encodes the rate-limiting subunit of human telomerase, is expressed at a high level in embryonic tissues and stem cells, but is repressed upon differentiation in the majority of adult somatic cells. Although the hTERT promoter has been studied extensively, the contribution of chromatin environment and the requirement of cis-elements have not been determined for the repression of the endogenous hTERT transcription. Previous results, including our own, have indicated that native chromatin plays a critical role in the regulation of hTERT transcription. hTERT transcription can be reversibly induced by inhibition of histone deacetylases and this induction is accompanied by chromatin remodeling at the hTERT promoter. Furthermore, we and others have shown that transiently transfected plasmid reporters are not ideal models for the repression of endogenous hTERT gene in somatic cells. Based on these findings, we hypothesize that chromatin environment is a critical component of the regulatory mechanisms for the repression of the native hTERT promoter. Here, we plan to study the molecular details of hTERT repression in a chromatin context using TPA-induced U937 cell differentiation as a model. We propose to pursue three interconnected aims: (1) To determine the role of global chromatin environment in hTERT repression during differentiation; (2) To determine sequential events of nuclear factor recruitment and histone modifications that occur at the hTERT core promoter region during differentiation. (3) To create a novel chromosome-based reporter system and dissect the roles of cis-regulatory elements in hTERT repression.